Photopolymer serum separator

ABSTRACT

Contemplated whole blood separators tubes include a curable composition with a density intermediate to the density of serum and the cell-containing fraction. After centrifugation, the curable composition is located between the serum and the cell-containing fraction and preferably irradiated with UV light to initiate a curing reaction in which the curable composition solidifies to a barrier that is immobilized and resistant to breakdown at freezing temperatures and extended storage.

This application is a continuation of U.S. patent application havingSer. No. 12/691,670 filed on Jan. 21, 2010, now allowed, which is acontinuation of U.S. patent application having Ser. No. 11/499,436 filedon Aug. 4, 2006, issued on Mar. 9, 2010 as U.S. Pat. No. 7,674,388,which claims the benefit of priority to U.S. provisional applicationSer. No. 60/707,299 filed on Aug. 10, 2005.

Field of The Invention

The field of the invention is serum separation, and especially to serumseparation using a polymerizable composition that forms a barrierbetween the separated phases.

BACKGROUND OF THE INVENTION

Among other sample manipulations, blood separation is a routine processthat is typically required in many analytic tests. Most commonly,separation is carried out in a centrifuge using a polymeric gel having adensity (about 1.04) that is between the heavier cell-containing phaseand the lighter serum-containing phase. Examples for separation devicesthat use intermediate density polymers are found in U.S. Pat. Nos.3,647,070 where polymer spheres form the barrier layer, while U.S. Pat.No. 5,266,199 describes a tube-and-ball valve that controls separationof the serum from the cell-containing phase. However, such barriers areoften either incomplete and tend to leak, or impracticable for variousreasons.

Alternatively, relatively impervious silicone-containing barrier layerscan be used in the serum separation as described in U.S. Pat. No. 3,780,935, and drug-impermeable separation polymers are described in EP 0 928301 in which fluid polymers are used as a barrier layers. Similarly,U.S. Pat. No. 4,235,725 describes the use of polybutadiene plus fillermaterial as a barrier forming material. Such barrier materials oftenprovide at least some advantage, but typically fail to maintain theseparation over a prolonged period. In still further known serumseparation devices, high-density polymers can be employed for bloodseparation in which the density is adjusted to a desirable degree with adensity reducing component as taught in EP 0 705 882. Such compositionsare often highly compatible with blood, and often exhibit favorableviscosity. However, such barrier layers are often unstable overprolonged periods. Alternatively, the viscosity of the intermediatepolymer may also be increased in the separator tube byphotopolymerization before the tubes is used for collection andseparation as described, for example, in U.S. Pat. No. 6,361,700 or U.S.Pat. No. 6,248,844. Once more, while such tubes often provide increasedsample stability, the barrier layer is typically unstable over prolongedperiods and will deteriorate upon freezing.

Still further known devices and methods are described in EP 0 520 185using fatty acid amides admixed with a gel, and EP 0 744 026 in which aperipheral water swellable band is taught as a barrier forming means.U.S. Pat. No. 3,920,557 described use of beads coated with an adhesiveto form a barrier layer between the serum and the cell containing phase,while U.S. Pat. No. 4,101,422 discloses copolyesters with specificmolecular weight and viscosity as barrier-forming compositions. Whilesuch known compositions tend to maintain the separation over at leastrelatively short periods, the separation layers are often notsufficiently stable to allow reliable storage over several days or whilefrozen.

Therefore, while numerous compositions and methods for centrifugal serumseparation are known in the art, all or almost all of them suffer fromone or more disadvantages. Thus, there is still a need to provideimproved composition and methods to improve serum separation devices.

SUMMARY OF THE INVENTION

The present invention is directed to devices and methods for serumseparator tubes that include a curable composition with an intermediatedensity to thereby locate between the serum fraction and thecell-containing fraction after centrifugation. Once separation of thephases is achieved, the curable composition is then subjected toconditions effective to solidify, crosslink, or otherwise strengthen thebarrier formed by the curable composition.

Therefore, in one aspect of the inventive subject matter, a serumseparator tube comprises a curable composition that is formulated tohave a density between an average density of a serum fraction of wholeblood and a cell-containing fraction of whole blood, and that isformulated to be mixable with or flowable in whole blood. Mosttypically, the composition will comprise a plurality of reactive groupsin an amount effective to form a crosslinked composition upon initiationof crosslinking, wherein the crosslinked composition is impermeable tothe cell-containing fraction of whole blood.

The curable composition in some preferred aspects is flowable andoptionally thixotropic and will most preferably comprise a polymer(e.g., polyisoprene, polyester, polyacrylate, and/or silicone oil). Suchpolymers will typically include a reactive group (e.g., as pendant orterminal group) to allow formation of the crosslinked composition usinga photo-initiated radical reaction. The photoinitiated reaction mayproceed directly without a photoinitiator, or may require a one or morephotoinitiators. Irradiation is preferably performed with UV light.Alternatively, it is contemplated that the crosslinked composition mayalso be crosslinked in a reaction using a radical starter (e.g.,chemically or thermally activated).

Consequently, in another aspect of the inventive subject matter, amethod of separating whole blood into a serum fraction and acell-containing fraction includes a step of providing a collection tubethat contains a curable composition and mixing whole blood with thecurable composition, wherein the curable composition has a densitybetween an average density of the serum fraction and the cell-containingfraction. The sample is then spun at a centrifugal force sufficient toseparate the serum fraction and the cell-containing fraction such thatthe curable composition forms a barrier layer between the serum fractionand the cell-containing fraction, and in yet another step, the curablecomposition is cured after centrifugation.

Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A is a schematic of a serum tube before centrifugation.

FIG. 1B is a schematic of the serum tube of FIG. 1 after centrifugation.

DETAILED DESCRIPTION

The inventor has discovered that serum separation can be significantlyimproved using a polymeric barrier material that is cured after thematerial has settled in a position between the lighter serum and theheavier cell-containing phase. Most preferably, it is contemplated thatthe remaining components and methods of serum separation remain ascurrently practiced.

In one exemplary aspect of the inventive subject matter, as shown inFIG. 1A, a serum separator tube 100 comprises an evacuated and sterileglass or plastic tube (e.g., similar or identical to VACUTAINER™ tubes(Franklin Lakes, NJ USA 07417)) having a removable top with a portionthat can be pierced to receive the blood 120. The curable material 110forming the barrier layer is preferably a biocompatible organic polymerwith a density of between about 1.04-1.06 g/cm³, and most preferably1.04 g/cm^(3.) Preferred polymers comprise a polyester backbone similarto those described in U.S. Pat. Nos. 6,361,700 and 6,248,844, both ofwhich are incorporated by reference herein. Polymerization is preferablycarried out to achieve the desired density of between about 1.04 - 1.06g/cm³. However, and in contrast to the methods and compositions providedin the '700 and '844 patent, polymerization is not run to completion butstopped using a polymerization terminator (e.g., using radicalquenchers, catalyst complexing agent, etc.) in a minimum amounteffective to stop further polymerization.

As the sample contacts the incompletely cured polymer (the curablecomposition), it is contemplated that the polymerization terminator isdiluted to a concentration that allows the polymerization to bere-initiated. Prior to re-initiation, the whole blood sample isseparated in the container by centrifugation as shown in FIG. 1B, whichwill leave the cell-containing fraction 125 in the bottom portion of thecontainer 100 and the serum fraction 123 in the upper portion of thecontainer, wherein both fractions are separated by the incompletelycured polymer 110. Most preferably, re-initiation of polymerization maybe assisted by irradiating the polymer with UV light or otherhigh-energy source. Thus, it should be appreciated that the polymeric isadditionally cured after the separation is completed and the soseparated serum can then be accessed without contamination of a pipette,decanted, or even frozen. Moreover, it should be recognized that thefinal cured barrier layer is substantially permanent (i.e., stable overseveral days, or even weeks).

While it is generally preferred that the separator tubes include apolyester polymer, it should be noted that the exact nature of thepolymeric material is not limiting to the inventive subject matter, andthat numerous alternative polymers are also suitable. Indeed all knownpolymers suitable for blood separation are deemed appropriate for useherein, including silicon oil, polyamides, olefinic polymers,polyacrylates polyesters and copolymers thereof, polysilanes, andpolyisoprenes. To achieve a desired initial density (typically betweenabout 1.03 and 1.05), it is contemplated that the density may beadjusted by virtue of molecular composition, as well as by inclusion ofappropriate filler material (e.g., silica, latex, or other inertmaterial). For example, suitable polymeric materials are described inU.S. Pat. Nos. 3,647,070, 3,920,557, or 3,780, 935, or in EP 0 928 301or 0 705 882, which are incorporated by reference herein. Furthermore,it is contemplated that the serum separators may include additionalmaterials and/or reagents to achieve a desired purpose. For example, theseparators presented herein may include EDTA, heparin, citrate,dextrose, etc. It should be noted that the term “serum” is used hereinto also include plasma, and other substantially cell free fluids derivedfrom whole blood.

Depending on the particular material, it is contemplated that the modeand/or mechanism of polymerization to the separator polymer may varyconsiderably, and all know manners of polymerization are deemed suitablefor use herein. For example, contemplated polymerizations includevarious radical or cationic polymerizations (e.g., using photolabilecompounds, radical startes, etc.), condensation polymerizations,esterifications, amide formation, etc. Thus, reactive groups willespecially include acid groups (and most preferably mono- anddicarboxylic groups), conjugated diene groups, aromatic vinyl groups,and alkyl(meth)acrylate. Such exemplary reactive groups and reactionconditions are described, for example, in U.S. Pat. No. 6,989,226, whichis incorporated by reference herein. It should furthermore beappreciated that the reactive groups can be coupled to the terminus of apolymer as end groups as described in WO 99/64931, which is incorporatedby reference herein, or that the reactive groups may be provided aspendant groups (e.g., as described in U.S. Pat. No. 5,336,736,incorporated by reference herein).

It is generally preferred that polymerization is fully supported byreactive groups on polymer, but additional reagents may also besuitable, including radical starters, including those described in U.S.Pat. No. 5,582,954, 4,894,315, and 4,460,675, which are incorporated byreference herein. Additionally contemplated compositions also includethose that provide a crosslinking group to the polymer such that thepolymer has reactive groups that react with a bifunctional crosslinker(e.g., ethylenically unsaturated compounds) to thereby form crosslinkedpolymers.

Thus, in another aspect of the inventive subject matter, a firstpolymerization may be performed to form the barrier material, and asecond polymerization reaction or curing reaction employs a reactionthat involves one or more reactive groups in the barrier polymer toharden/solidify the barrier. For example, where the barrier polymer is apolyester, curing of the polymer may be performed using a radicalpolymerization reaction that includes peroxo-, photolabile, or redoxstarters to generate a radical species that initiates the curingreaction between reactive groups (ethylenically unsaturated groups,epoxy groups, etc.) of polymeric strands of the polyester.Alternatively, catalyzed CLICK chemistry or other reactions may beemployed so long as such reactions do not interfere with a downstreamanalytic process, and/or so long as the barrier polymer is curable to adegree that allows decanting and/or freezing without loss of separation.

In other aspects of the inventive subject matter, the polymerizationreaction to form the barrier polymer may be incomplete (e.g., byaddition of polymerization terminator, or depletion of reactivesubstrate) and the so formed barrier material can be cured after thebarrier has formed by re-initiation of proper reaction condition. Forexample, where the concentration of the polymerization terminator hasfallen below an inhibitory level via dilution with blood, polymerizationmay be started by UV irradiation. Alternatively, and depending on theparticular curing mechanism, addition of reactive materials and/orcrosslinkers may also be suitable. Further polymers and reactive groupssuitable for photopolymerization are described in U.S. Pat. No.5,086,138, which is incorporated by reference herein.

Thus, specific embodiments and applications of curable serum separatorbarrier materials have been disclosed. It should be apparent, however,to those skilled in the art that many more modifications besides thosealready described are possible without departing from the inventiveconcepts herein. The inventive subject matter, therefore, is not to berestricted except in the spirit of the appended claims. Moreover, ininterpreting both the specification and the claims, all terms should beinterpreted in the broadest possible manner consistent with the context.In particular, the terms “comprises” and “comprising” should beinterpreted as referring to elements, components, or steps in anon-exclusive manner, indicating that the referenced elements,components, or steps may be present, or utilized, or combined with otherelements, components, or steps that are not expressly referenced.Furthermore, where a definition or use of a term in a reference, whichis incorporated by reference herein is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply.

1. A separation container containing a separator composition comprising:a curable composition disposed within the container, and that isformulated to have a density between an average density of a lighterphase of a fluid and a heavier phase of the fluid, and that is furtherformulated to be mixable with or flowable with the fluid; wherein thecurable composition comprises a polymerizable compound having aplurality of reactive groups in an amount effective to form a solidcrosslinked composition upon initiation of crosslinking through curingthe curable composition; and wherein the curable composition forms abarrier between the phases due to separation, and upon curing afterseparation forms the crosslinked composition in a manner where thebarrier becomes solid and impermeable to the phases.
 2. The container ofclaim 1, wherein the curable composition comprises an incompletely curedpolymer.
 3. The container of claim 2, wherein the solid barriercomprises the crosslinked composition formed from curing theincompletely cured polymer.
 4. The container of claim 1, wherein thecured barrier is immobilized within the container.
 5. The container ofclaim 1, wherein the curable composition comprises a polymerizationterminator.
 6. The container of claim 5, wherein a concentration of thepolymerization terminator falls below an inhibitory level via dilutionwith the fluid.
 7. The container of claim 1, wherein the barrier retainsseparation between the phases after freezing.
 8. The container of claim1, wherein the fluid comprises blood.
 9. A method of separating a fluidinto a light fraction and heavy fraction using a separation containerand a separator composition, comprising: providing a container thatcontains a curable composition, the curable composition formulated tohave a density between an average density of a light fraction and aheavy fraction of a fluid, and comprises a polymerizable compound havinga plurality of reactive groups in an amount effective to form a solidcrosslinked composition upon initiation of crosslinking through curingthe curable compound placing the fluid in the container with the curablecompound; separating the sample at a force sufficient to separate thelight fraction and the heavy fraction such that the curable compositionforms a barrier layer between the separated light fraction and heavyfraction of the fluid; and curing the curable composition after the stepof centrifuging causing the barrier to become solid through forming thesolid crosslinked composition.
 10. The method of claim 9, wherein thecurable composition comprises an incompletely cured polymer.
 11. Themethod of claim 10, wherein the step of curing the curable compoundincludes re-initiating curing of the incompletely cured polymer to forthe solid barrier.
 12. The method of claim 9, wherein the step of curingincludes immobilizing the solid barrier within the container.
 13. Themethod of claim 9, wherein the curable composition comprises apolymerization terminator.
 14. The method of claim 13, wherein the stepof placing the fluid in the container includes diluting a concentrationof the polymerization terminator to fall below an inhibitory level. 15.The method of claim 9, further comprising allowing the barrier to retainseparation between the phases after freezing.
 16. The method of claim 9,wherein the step of placing fluid into the container includes usingblood as the fluid.